DHX32 And FLIP Expression And Their Clinical Significance In Colorectal Carcinoma

Objective①To develop fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) for quantitating DEAH (Asp-Glu-Ala-His) box polypeptide 32 (DHX32) mRNA, FLICE-like inhibitory protein long form (FLIP(L)) mRNA and FLICE-like inhibitory protein short form (FLIP(S)) mRNA.②To explore the expression and the clinical significance of DHX32 and FLIP in colorectal carcinoma.③To clarify the relationship between DHX32 and FLIP, and to explore their possible function in colorectal carcinoma development and progression.Methods①The specific primers and TaqMan probe of DHX32, FLIP(L) and FLIP(S) were designed using primer express 2.0 according to mRNA sequence and the principle of TaqMan probe quantitative assay.②Optimize the reaction condition and take the technology evaluation to develop FQ-RT-PCR for relatively quantitating DHX32 mRNA, FLIP(L) mRNA and FLIP(S) mRNA.③The total RNA isolated from human colorectal carcinoma tissue and adjacent colorectal tissue was reversely transcribed into cDNA. Housekeeping geneβ-actin was used as internal reference. The FQ-PCR method was used to measure cDNA to analyze the expression of DHX32, FLIP(L) and FLIP(S).④The defferent expression of DHX32 and FLIP was linked with the clinical indicator.Results①FQ-RT-PCR for relative quantitating DHX32 mRNA was successfully established. The efficiency of PCR was 0.85. The specificity and the repetition were great (the intra-assay and inter-assay coefficient variation was 1.85% and 2.95%).②DHX32 mRNA was detectable in 75% of tumor tissue, 26.4% of adjacent tissue. The expression of DHX32 in colorectal carcinoma tissue was higher than in adjacent colorectal tissue (P<0.05).③The expression of DHX32 in colorectal carcinoma was related to the histological grade, the DUKES clinical stage, carcino-epistom and the lymphatic follicle diversion (P<0.05). The more malignancy, the higher the expression of DHX32.④FQ-RT-PCR for relative quantitating FLIP(L) and FLIP(S) was successfully established. The efficiency of PCR was 0.89 and 0.88. The specificity and the repetition were great (the intra-assay and inter-assay coefficient variation were less than 3%).⑤FLIP mRNA was detectable in all of tumor and adjacent tissue. The expression of FLIP in colorectal carcinoma was higher than in adjacent colorectal tissue (P<0.05).⑥The expression of FLIP in colorectal carcinoma was related to the histological grade (P<0.05). The histological grade is lower; the expression level of FLIP is higher.⑦The expression of DHX32 was related to the expression of FLIP in colorectal carcinoma (P<0.01), r =-0.597.⑧The expression of DHX32 was related to the rate of FLIP(L)/FLIP(S) in colorectal carcinoma (P=0.025), r =-0.326.Conclusions①FQ-RT-PCR for quantitating DHX32, FLIP(L) and FLIP(S) was a sensitive, specific, rapid and stable method and a useful technique. ②The expression of DHX32 was high and related to the histological grade, the DUKES clinical stage, tumor thrombus and the lymphatic follicle diversion in colorectal carcinoma, it indicated that DHX32 maybe a reference guideline for malignant in colorectal carcinoma.③The expression of FLIP was high and related to the histological grade in colorectal carcinoma. It indicated that FLIP might play a crucial role in the carcinogenesis of colorectal carcinoma by blocking the receptor-mediated apoptotic pathway.④DHX32 closely correlates with FLIP each other. They might play an important role in the carcinogenesis of colorectal carcinoma.